Bifidobacterium in the treatment of inflammatory disease

ABSTRACT

A strain of  Bifidobacterium  isolated from resected and washed human gastrointestinal tract is significantly immunomodulatory following oral consumption in humans. The strain is useful in the prophylaxis and/or treatment of undesirable inflammatory activity, especially gastrointestinal inflammatory activity such as inflammatory bowel disease or irritable bowel syndrome. The inflammatory activity may also be due to cancer.

INTRODUCTION

This invention relates to probiotic Bifidobacterium strains which havevarious applications in foodstuffs and in medicine. More particularly,the invention relates to probiotic strains of bifidobacteria which arecapable of beneficially modifying and consequently alleviatingobservable symptoms in inflammatory disease.

Consumers are becoming increasingly aware of matters which may benecessary for maintenance of their environment, health and nutrition. Inresponse, scientific research has focused upon the roles that diet,stress, and modern medical practices (e.g. antibiotics and radiotherapy)may play in threatening human health. In particular, population dynamicsshifting towards older societies are increasing the incidence ofillnesses which may be caused by deficient or compromised microflorasuch as gastrointestinal tract (GIT) infections, constipation, irritablebowel syndrome (IBS), inflammatory bowel disease (IBD)—Crohn's diseaseand ulcerative colitis, food allergies, antibiotic-induced diarrhoea,cardiovascular disease, and certain cancers (e.g. colorectal cancer).

Probiotics have been defined as live microbial food supplements whichbeneficially affect the host by improving the intestinal microbialbalance, or more broadly, as living micro-organisms, which uponingestion in certain numbers, exert health effects beyond inherent basicnutrition. Cocktails of various micro-organisms, particularly species ofLactobacillus and Streptococcus, have traditionally been used infermented dairy products to promote health.

In recent years the commercial manufacture and marketing of functionalfoods (foods which affect functions of the body in a targeted manner soas to bring about positive affects on physiology and nutrition),particularly probiotic (Acidophilus-Bifidus) yoghurts, has spread fromthe well-established Japanese niche market place into the lucrative andexpanding European Union. While a number of probiotic bacteria of humanorigin are now being exploited commercially (e.g., L. acidophilus LA-1),many consumers, consumer organisations, and members of the scientificcommunity are sceptical of such products and their publicised probioticclaims. The diary-food industry is therefore under considerable pressureto scientifically validate these new probiotic food products.

Criteria which have been suggested for the selection of potentiallyeffective probiotic micro-organisms may be summarised as follows: humanorigin, non-pathogenic behaviour, resistance to technological processes(i.e., viability and activity in delivery vehicles), resistance togastric acidity and bile toxicity, adhesion to gut epithelial tissue,ability to colonise the GIT, production of antimicrobial substances,ability to modulate immune responses, and the ability to influencemetabolic activities (e.g., cholesterol assimilation, lactase activity,vitamin production) (Huis in't Veld J, Shortt C. Selection criteria forprobiotic micro-organisms. In: Leeds, A. R., Rowland, I. R. eds. GutFora and Health—Past, Present and Future. London: The Royal Society ofMedicine Press Ltd., 1996:19-26).

Bifidobacteria are one of several predominant culturable bacteriapresent in the colonic microflora.

The functions of endogenous bifidobacteria in the colon have not beencompletely elucidated. However it is recognised that exclusivelybreast-fed infants have a reduced risk of diarrhoea compared withformula-fed infants. The fact that these infants have greater numbers ofcolonic bifidobacteria may in part explain this observed healthadvantage as the occupation of available niches in the GIT by largenumbers of nonpathogenic bifidobacteria may help prevent bacterialinfection. The pathogenesis of Crohn's disease is thought to be relatedto colonic bacterial microflora (Targan, S. and Shanahan, F.Inflammatory bowel disease: From bench to bedside. Williams and Wilkins1994.) It has recently been found that patients suffering from activeCrohn's disease have significantly less recoverable bifidobacteria intheir faeces compared with healthy individuals. This reduction inbifidobacteria numbers was observed to be directly correlated withdecreased levels of β-D galactosidase production and activity (Favier,C. et al; Dig. Dis. Sci. 1997;42:817-822). β-D galactosidase is anenzyme produced by bifidobacteria. These results support suggestionsproposed in other studies that strains of bifidobacteria may playimportant roles in maintaining a balanced healthy intestinal microflora.

Bifidobacteria are considered to be probiotics as they are livingorganisms which exert healthy effects beyond basic nutrition wheningested in sufficient numbers. Numerous ingested bifidobacteria mustreach the site of action in the gut in order to exert a probioticeffect. A minimum level of approximately 10⁶-10⁷ viable bifidobacteriaper gram intestinal contents has been suggested (Bouhnik, Y., Lait1993.: 73:241-247). There are reports in the literature which show thatin vivo studies completed in adults and in infants indicate that somestrains of bifidobacteria are capable of surviving passage through thegastrointestinal tract. Significant differences have been observedbetween the abilities of different bifidobacteria strains to tolerateacid and bile salts, indicating that survival is an important criterionfor the selection of potential probiotic strains.

Ingestion of bifidobacteria can improve gastrointestinal transit.

Furthermore, indirect evidence in humans demonstrates that consumingmilk fermented by bifidobacteria can lead to reduced levels of certainfaecal enzymes such as β-D galactosidase implicated in the conversion ofprocarcinogens to carcinogens (Bouhnik Y. et al; Eur. J. Clin. Nutr.1996;50:269-273). Faecal-borne putrefaction metabolities such asp-cresol, indole and ammonia were also reduced when subjects consumedmilk fermented by Bifidobacterium longum and S. thermophilus (Takiguchi,R. et al. Bifidus—Flores, Fructus et Semina 1996;9:135-140).

Antimicrobial activity has been reported to be associated withbifidobacteria. Also, bifidobacteria have been shown to modulate variousparameters of the immune system.

Mucosal inflammation in IL-10 deficient mice has been reported to bereduced by feeding the subject animals a preparation of lactic acidbacteria (Madsen, K. et al. Gastroenterol. 1997;112:A1030.). Furtherstudies completed in rats have demonstrated that ingestion ofbifidobacteria can suppress aberrant crypt foci (early preneoplasticlesions) formation in the colon (Kulkarni, N. and Reddy, B. Proc. Soc.Experim. Biol. Med. 1994; 207;278-283.) in addition to significantdecreases in colon tumor incidence and in the numbers of tumors present(Singh, J. et al Carcinogenesis 1997;18:833-841).

There is an on-going search for probiotic strains with particularbeneficial effects on nutrition and therapy and on health generally.

STATEMENTS OF INVENTION

The invention provides a strain of Bifidobacterium isolated fromresected and washed human gastrointestinal tract which is significantlyimmunomodulatory following oral consumption in humans.

The strain of Bifidobacterium preferably effects changes in animmunological marker when introduced into a system comprising cellswhich interact with the immune system and cells of the immune system.Preferably the cells which interact with the immune system areepithelial cells. Preferably the immunological marker is a cytokine,especially TNFα.

In a preferred embodiment the cells which interact with the immunesystem and the immune system cells are of matched origin.

The cells which interact with the immune system are of gastrointestinal,respiratory or genitourinary origin.

The cells of the immune system are preferably of gastrointestinal,respiratory or genitourinary origin.

The invention also provides a strain of Bifidobacterium longum infantisisolated from resected and washed human gastrointestinal tract which issignificantly immunomodulatory following oral consumption in humans.

The strain of Bifidobacterium which has significant anti-inflammatoryeffect following oral consumption in humans.

The strain of Bifidobacterium is preferably isolated from resected andwashed human gastrointestinal tract which is capable of combating theeffects of inflammatory bowel disease, said capability being maintainedin the presence of physiological concentrations of human bile and humangastric juice. The capability of combating the effects of inflammatorybowel disease is measured by measuring a reversal of a wasting diseaseinduced in severe combined immunodeficient recipient mice (SCID) whichhave been administered purified CD4⁺, CD45RB^(high) T cells.

The capability of the strain of Bifidobacterium longum infantis tocombat the effects of inflammatory bowel disease can also be measured bymeasuring the reduction in colonic inflammation in IL-10 deficient mice(IL-10⁺129 Svex strain) following administration of one or more of thestrains of Bifidobacterium longum infantis according to the inventionalone or in combination with a strain of Lactobacillus salivarius ashereinafter defined.

Interleukin 10 (IL-10) is an important regulatory cytokine thatsupresses effector functions of macrophage/monocytes, T helper 1 (Th1)cells, and natural killer cells. In addition, IL-10 augmentsproliferation and differentiation of B cells. Murine models lacking theIL-10 gene spontaneously develop inflammatory bowel disease andgastrointestinal tumors. The gastrointestinal flora have been implicatedin the pathogenesis of these disease states as germ free animals do notdevelop disease.

The strain of Bifidobacterium preferably has inhibitory activity againsta broad range of Gram positive and Gram negative bacteria.

Preferably the strain of Bifidobacterium exhibits a broad-spectrum ofactivity against bacteria including Staphylococcus, Pseudomonas,Coliform and Bacillus species.

In a particular aspect the invention provides strain of Bifidobacteriumlongum infantis UCC35624 or mutant or variant thereof.

A deposit of Bifidobacterium longum infantis strain UCC 35624 was madeat the National Collections of Industrial and Marine Bacteria Limited(NCIMB) on Jan. 13, 1999 and accorded the accession number NCIMB 41003.

In one embodiment the mutant is a genetically modified mutant.

In one embodiment the variant is a naturally occurring variant ofBifidobacterium longum infantis UCC35624.

The strain of Bifidobacterium may be in the form of viable cells.Alternatively the strain of Bifidobacterium is in the form of non-viablecells.

The invention also provides an antimicrobial agent obtained from astrain of Bifidobacterium of the invention which is antagonistic to thegrowth of other organisms.

In a further aspect the invention provides a formulation which comprisesa strain of Bifidobacterium of the invention.

The formulation may comprise two or more strains of Bifidobacterium.

The formulation may include another probiotic material. Alternatively oradditionally the formulation includes a prebiotic material.

The formulation may which include a strain of Lactobacillus salivarius.

The strain of Lactobacillus salivarius may be in the form of viablecells or in the form of non-viable cells.

The Lactobacillus salivarius is preferably isolated from resected andwashed human gastrointestinal tract, the Lactobacillus salivarius beingsignificantly immunomodulatory following oral consumption in humans.Preferably the strain of Lactobacillus salivarius is isolated fromresected and washed human gastrointestinal tract which inhibits a broadrange of Gram positive and Gram negative micro-organisms.

In a preferred embodiment the strain of Lactobacillus salivariussecretes a product having antimicrobial activity into a cell-freesupernatant, said activity being produced only by growing cells andbeing destroyed by proteinase K and pronase E, the inhibitory propertiesof said strain and its secretory products being maintained in thepresence of physiological concentration of human bile and human gastricjuice.

Such strains of Lactobacillus salivarius are disclosed in WO 98/35014.

Ideally the strain of Lactobacillus salivarius is Lactobacillussalivarius strain UCC 118 or a mutant or variant thereof. The mutant isa genetically modified mutant. The variant may be a naturally occurringvariant of Lactobacillus salivarius.

A deposit of Lactobacillus salivarius strain UCC 118 was made at theNCIMB on Nov. 27, 1996 and accorded the accession number NCIMB 40829.

Preferably the formulation includes an ingestable carrier. Theingestable carrier may be a pharmaceutically acceptable carrier such asa capsule, tablet or powder.

The ingestable carrier may be a food product such as acidified milk,yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spreads,dressings or beverages.

The formulation may comprise a protein and/or peptide, in particularproteins and/or peptides that are rich in glutamine/glutamate, a lipid,a carbohydrate, a vitamin, mineral and/or trace element.

In one embodiment the Bifidobacterium is present at more than 10⁶ cfuper gram of delivery system.

In another embodiment the formulation includes an adjuvant.

The formulation may include a bacterial component. The formulation mayalternatively or additionally include a drug entity. The formulation mayalso include a biological compound.

The formulation may be in a form for oral immunisation.

The invention further provides a strain of Bifidobacterium or aformulation thereof for use in foodstuffs.

In another aspect the invention provides a strain of Bifidobacterium ora formulation thereof for use as a medicament.

The strain or formulation may be for use in the prophylaxis and/ortreatment of undesirable inflammatory activity.

The strain or formulation may be for use in the prophylaxis and/ortreatment of undesirable gastrointestinal inflammatory activity such asinflammatory bowel disease eg. Crohns disease or ulcerative colitis,irritable bowel syndrome, pouchitis or post infection colitis.

The undesirable inflammatory activity may be due to cancer.

In addition the strain or formulation may be for use in the prophylaxisand/or treatment of gastrointestinal cancer(s).

The strain or formulation may be used for the prophylaxis of cancer.Further, the strain or formulation may be for use in the prophylaxisand/or treatment of systemic disease such as rheumatoid arthritis.

The strain or formulation may be for use in the prophylaxis and/ortreatment of autoimmune disorders due to undesirable inflammatoryactivity.

The strain or formulation may be for use in the prophylaxis and/ortreatment of cancer due to undesirable inflammatory activity.

The strain or formulation may be for use in the prophylaxis and/ortreatment of diarrhoeal disease due undesirable inflammatory activity,such as Clostridium difficile associated diarrhoea, Rotavirus associateddiarrhoea or post infective diarrhoea.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 is a graph of cfu/ml versus time for Bifidobacterium longuminfantis strain 35612 as described in Example 2;

FIG. 2 is a graph of cfu/ml versus time for Bifidobacterium longuminfantis strain 35624 as described in Example 2;

FIG. 3 is a graph of percentage weight change versus time (days) forfive SCID mice (1-5) administered strain UCC 35624 as described inExample 5;

FIG. 4 is a graph of average percentage weight change versus time (days)for the SCID mice (1-5) administered strain UCC 35624 as described inExample 5;

FIG. 5 is a graph of percentage weight change versus time (days) formice (6-10) administered a combination of strains Lactobacillussalivarius UCC 118 and UCC 35624 as described in Example 5;

FIG. 6 is a graph of average percentage weight change versus time (days)for mice (6-10) administered a combination of strains UCC 118 and UCC35624 as described in Example 5;

FIG. 7 is a graph of percentage weight change versus time (days) formice (11-15) administered a combination of stains UCC 118 and UCC 35624as described in Example 5;

FIG. 8 is a graph of average percentage weight change versus time (days)for mice (11-15) administered a combination of strains UCC 118 and UCC35624 as described in Example 5;

FIG. 9 is a bar chart of TNFα levels in patient and control samples inthe presence of PBMCs and Bifidobacteria longum infantis as described inExample 7;

FIG. 10 is a bar chart showing TNFα and IL-8 levels in co-cultures ofepithelial cells, PBMCs and Bifidobacterium longum infantis as describedin Example 7. Controls represent co-cultures of epithelial cells andPBMCs alone;

FIG. 11 are bar charts of peripheral blood cytokine levels followingconsumption of Bifidobacterium longum infantis by healthy humanvolunteers (n=18) for three weeks as described in Example 8;

FIG. 12 are bar charts of serum levels of TNFα and IL-1RA followingconsumption of Bifidobacterium longum infantis to healthy humanvolunteers (n=18) as described in Example 8;

FIG. 13 is a bar chart of TNFα levels in cell-free spent culturesupernatant of Bifidobacterium longum infantis and an MRS control asdescribed in Example 9;

FIG. 14 is a diagrammatic representation of a SCID mouse lower intestineafter treatment with Bifidobacterium longum infantis; and

FIG. 15 is a diagrammatic representation of the lower intestine of anuntreated SCID mouse.

DETAILED DESCRIPTION

We have isolated strains of probiotic bacteria which are capable ofbeneficially modifying and consequently alleviating observable symptomsin inflammatory disorders. These strains and the formulations preparedmay be used in a variety of foodstuffs and medicaments to combat theeffect of inflammatory disorders.

In vivo and in vitro studies were carried out using the probioticbacteria strains. It was found that humans fed with yoghurt containingBifidobacterium longum infantis UCC35624 show marked decreases in theirsystemic levels of IL-8. This strain may therefore have potentialapplication in the treatment of a range of inflammatory disorders,particularly if used in combination with current anti-inflammatorytherapies, such as non-steroid anti-inflammatory drugs (NSAIDs) orInfliximab.

The consumption of Bifidobacterium longum infantis by SCID mice was alsoexamined. While this experiment significantly attenuated inflammatoryactivity, mice consuming Bifidobacterium longum infantis retained solidstools while control mice suffered from diarrhoea. This anti-diarrhoealeffect could be related to the anti-inflammatory activity of thisinvention, possibly mediated via cAMP modulation.

It is unknown whether intact bacteria are required to exert ananti-inflammatory effect or if individual active components of theinvention can be utilised alone. Proinflammatory components of certainbacterial strains have been identified. The proinflammatory effects ofgram-negative bacteria are mediated by lipopolysaccharide (LPS). LPSalone induces a proinflammatory network, partially due to LPS binding tothe CD14 receptor on monocytes. It is assumed that components ofprobiotic bacteria possess anti-inflammatory activity, due to theeffects of the whole cell. Upon isolation of these components,pharmaceutical grade manipulation is anticipated.

The general use of Bifidobacterium longum infantis UCC35624 is in theform of viable cells. However, it can also be extended to non-viablecells such as killed cultures or compositions containing beneficialfactors expressed by Bifidobacterium longum infantis UCC35624. Thiscould include thermally killed micro-organisms or micro-organisms killedby exposure to altered pH or subjection to pressure. With non-viablecells product preparation is simpler, cells may be incorporated easilyinto pharmaceuticals and storage requirements are much less limited thanviable cells. Lactobacillus casei YIT 9018 offers an example of theeffective use of heat killed cells as a method for the treatment and/orprevention of tumour growth as described in U.S. Pat. No. 4,347,240.

The invention will be more clearly understood from the followingExamples.

EXAMPLE 1 Isolation of Probiotic Bacteria

Appendices and sections of the large and small intestine of the humanG.I.T., obtained during reconstructive surgery, were screened forprobiotic bacterial strains as shown in Table 1. TABLE 1Gastrointestinal tract tissue samples screened for the presence ofprobiotic bacteria Sample Location A Ileum B Colon C Ileal-caecal regionD Appendix E Appendix F Ileum G Ileal-caecal region

All samples were stored immediately after surgery at −80° C. in sterilecontainers. Frozen tissues were thawed, weighed and placed incysteinated (0.05%) one quarter strength Ringers' solution. Each samplewas gently shaken to remove loosely adhering microorganisms (termed—wash‘W’). Following transfer to a second volume of Ringers' solution, thesample was vortexed for 7 min to remove tightly adhering bacteria(termed—Sample ‘S’). In order to isolate tissue embedded bacteria,samples A, B and C were also homogenised in a Braun blender(termed—homogenate ‘H’). The solutions were serially diluted (dilution10⁻¹ from a wash sample was labelled W1, dilution 10⁻² was labelled W2and the same labelling system was used for the ‘S’ and ‘H’ samples) andspread-plated (100 μl) on to the following agar media: RCM (reinforcedclostridial media) and RCM adjusted to pH 5.5 using acetic acid; TPY(trypticase, peptone and yeast extract), Chevalier, P. et al. (1990) J.Appl. Bacteriol 68, 619-624). MRS (deMann, Rogosa and Sharpe); ROG(acetate medium (SL) of Rogosa); LLA (Liver-lactose agar of Lapiere);BHI (brain heart infusion agar); LBS (Lactobacillus selective agar) andTSAYE (tryptone soya agar supplemented with 0.6% yeast extract). Allagar media was supplied by Oxoid Chemicals with the exception of TPYagar. Plates were incubated in anaerobic jars (BBL, Oxoid) using CO₂generating kits (Anaerocult A, Merck) for 2-5 days at 37° C.

Gram positive, catalase negative rod-shaped or bifurcated/pleomorphicbacteria isolates were streaked for purity on to complex non-selectivemedia (TPY). Isolates were routinely cultivated in TPY medium unlessotherwise stated at 37° C. under anaerobic conditions. PresumptiveBifidobacteria species were stocked in 40% glycerol and stored at −20°and −80° C.

Fermentation End-Product Analysis

Metabolism of the carbohydrate glucose and the subsequent organic acidend-products were examined using an LKB Bromma, Aminex HPX-87H HighPerformance Liquid Chromatography (HPLC) column. The column wasmaintained at 60° C. with a flow rate of 0.6 ml/min (constant pressure).The HPLC buffer used was 0.01 N H₂SO₄. Prior to analysis, the column wascalibrated using 10 mM citrate, 10 mM glucose, 20 mM lactate and 10 mMacetate as standards. Cultures were propagated in modified MRS broth for1-2 days at 37° C. anaerobically. Following centrifugation for 10 min at14,000 g, the supernatant was diluted 1:5 with HPLC buffer and 200 μlwas analysed in the HPLC. All supernatants were analysed in duplicate.

Biochemical and Physiological Characterisation

Biochemical and physiological traits of the bacterial isolates weredetermined to aid identification. Nitrate reduction, indole formationand expression of β-galactosidase activity were assayed. Growth at both15° C. and 45° C. and protease activity on gelatin were determined.Growth characteristics of the strains in litmus milk were also assessed.

Antibiotic Sensitivity Profiles

Antibiotic sensitivity profiles of the isolates were determined usingthe ‘disc susceptibility’ assay. Cultures were grown up in theappropriate broth medium for 24-48 h, spread-plated (100 μl) onto agarmedia and discs containing known concentrations of the antibiotics wereplaced onto the agar. Strains were examined for antibiotic sensitivityafter 1-2 days incubation at 37° C. under anaerobic conditions. Strainswere considered sensitive if zones of inhibition of 1 mm or greater wereseen.

Isolation of Bifidobacteria sp.

Seven tissue sections taken from the human G.I.T. were screened for thepresence of strains belonging to the Bifidobacterium genus. There wassome variation between tissue samples as follows. Samples A (ileum) andE (appendix) had the lowest counts with approximately 10² cells isolatedper gram of tissue. In comparison, greater than 10³ cfu/g tissue wererecovered from the other samples. Similar numbers of bacteria wereisolated during the ‘wash’ and ‘sample’ steps with slightly highercounts in the ‘sample’ solutions of F (ileum) and G (ileal-caecal). Ofthose screened for tightly-adhering bacteria (homogenised), C(ileal-caecal) was the only tissue section that gave significant counts.

During the screening of some tissue sections, for example C and B, therewas not a direct correlation between counts obtained during a dilutionseries. This would indicate that some growth factors, either blood ortissue derived were being provided for the growth of the fastidiousbacteria in the initial suspension which was subsequently diluted out.

Strain Selection and Characterisation

Approximately fifteen hundred catalase negative bacterial isolates fromdifferent samples were chosen and characterised in terms of their Gramreaction, cell size and morphology, growth at 15° C. and 45° C. andfermentation end-products from glucose. Greater than sixty percent ofthe isolates tested were Gram positive, homofermentative cocci arrangedeither in tetrads, chains or bunches. Eighteen percent of the isolateswere Gram negative rods and, heterofermentative coccobacilli.

The remaining isolates (twenty-two percent) were predominantlyhomofermentative coccobacilli. Thirty eight strains were characterisedin more detail—13 isolates from G; 4 from F; 8 from D; 9 from C; 3 fromB and 1 from E. All thirty eight isolates tested negative both fornitrate reduction and production of indole from tryptophan.

Antibiotic Sensitivity Profiles

Antibiotics of human clinical importance were used to ascertain thesensitivity profiles of selected bifidobacteria. The bifidobacteriatested were sensitive to ampicillin, amoxycillin ceftaxime, ceftriaxone,ciprofloxacin, cephradine, rifampicin, amikacin, gentamicin andchloramphenicol. They were also resistant to netilmicin, trimethoprim,nalidixic acid, cefuroxime, vancomycin and tetracycline.

EXAMPLE 2 Acid Resistance

The first line of host defence that a micro-organism reaches followinghuman consumption is gastric acid in the stomach. A key factorinfluencing bacteria is survival in gastric juice. The survival andgrowth of Bifidobacterium longum infantis strains 35612 and 35624 in alow pH environment were examined. The strains were routinely cultured intrypticase-peptone-yeast extract (TPY) medium at 37° C. under strictanaerobic conditions (BBL Gas jars using the Merck Anaerocult A gas paksystem) for 12-24 h. Human gastric juice was obtained from healthysubjects by aspiration through a nasogastric tube (Mercy Hospital, Cork,Ireland). It was immediately centrigued at 13,000 g for 30 min. toremove all solid-particles, sterilised through 0.451 μm filters and 0.21μm filters and stored at 4° C. The pH and pepsin activity were measuredprior to experimental use. Pepsin activity was measured using thequantitative haemoglobin assay (Guantam, S. and R. S. de la Motte. 1989.Proteolytic enzymes, a practical approach. Chapter 3. R. J. Beynon andJ. S. Bond(eds.), IRL Press, Oxford University Press; Dawson, R. M.1969. pH and buffers. In Data for Biochemical Research p 138. R. M.Dawson, D. C. Elliot and K. M. Jones(eds.), Clarendon Press, Oxford).Survival of the strains at low pH in vitro was investigated using thefollowing assays:

(a) Cells were harvested from fresh overnight cultures, washed twice inphosphate buffer (pH 6.5) and resuspended in MRS broth adjusted to pH3.5, 3.0, 2.5 and 2.0 (with 1 N HCl) to a final concentration ofapproximately 10⁶ cfu/ml. Cells were incubated at 37° C. and survivalmeasured at intervals of 5, 30, 60 and 120 min. using the plate countmethod.

The strains survived with no loss of viability at pH 3.5. At pH 2.5there was a 3 log reduction over the 60 min. incubation period asdepicted in FIGS. 1 and 2.

Survival of Strains of Bifidobacterium in Human Gastric Juice

Fresh overnight cultures were harvested, washed twice in buffer (pH 6.5)and resuspended in human gastric juice to a final concentration of 10⁶cfu/ml. Survival was monitored over a 30-60 min incubation period at 37°C. The experiment was performed using gastric juice at pH 1.2(unadjusted) and pH 2.0 and 2.5 (adjusted using 1N NaOH).

Survival of the strains was increased in gastric juice at pH 2.0, whencompared with gastric juice at pH 1.2. After 30 min incubation no viablecells were recovered at either pH as shown in Table 2. TABLE 2 Survivalof Bifidobacterium sp. in human gastric juice* TIME (min) STRAIN pH 0 530 60 35612 1.2 7.56 0.00 0.00 0.00 2.0 6.27 6.31 2.88 0.00 35624 1.25.96 4.18 0.00 0.00 2.0 6.33 6.32 0.00 0.00 35652 1.2 6.16 3.78 0.000.00 2.0 8.45 8.40 3.45 0.00 35648 1.2 6.00 0.00 0.00 0.00 2.0 7.89 6.450.00 0.00 35687 1.2 6.68 0.00 0.00 0.00 2.0 8.75 8.77 3.34 0.00 BO 2.08.41 8.56 8.42 8.43 10 2.0 8.39 8.56 4.64 0.00 6.3 2.0 8.75 8.75 8.298.42 B. longum 6 2.0 8.15 8.02 0.00 0.00*survival expressed as log₁₀ cfu/ml

EXAMPLE 3 Bile Resistance

In the evaluation of the effectiveness of using lactic acid bacteria asbeneficial members of the gastrointestinal tract, it is considered thatresistance to bile acids is an important biological straincharacteristic required for survival in this hostile environment and inaddition they must not impinge on the health of the host by producingtoxic compounds such as deoxycholic (DCA) and lithocholic acid (LCA)which have been implicated in a number of cytotoxic phenomena.

A number of Bifidobacterium longum infantis strains were streaked ontoTPY agar plates supplemented with porcine bile (B-8631, Sigma ChemicalCo. ltd., Poole) at concentrations of 0.3, 0.5, 1.0, 1.5, 5.0 and 7.5%(w/v) (Legrand-Defretin, R. et al., Lipids 1991; 26 (8), 578-583).Porcine bile is the closest in composition to human bile with respect tobile salt/cholesterol and phospholipid/cholesterol ratios. Plates wereincubated at 37° C. under anaerobic conditions and growth was recordedafter 24-48 h. Strain 35624 was found to be strongly bile resistant andgrew to confluence at up to 55 porcine bile as shown in Table 3. TABLE 3Growth of Bifidobacterium sp. isolates in the presence of porcine bile %(w/v) PORCINE BILE STRAIN 0.0 0.3 0.5 1.0 1.5 5.0 7.5 34612 + − − − − −− 35624 + + + + + + − 35652 + − − − − − − 35658 + + + + − − − 35687 + −− − − − −−, no growth;+, confluent growth

Human bile was obtained from several human gall bladders and sterilisedat 80° C. for 10 min. The bile acid composition of human bile wasdetermined using reverse phase High Performance Liquid Chromatography(HPLC) in combination with a pulsed amperometric detector according tothe method of Dekcker, R. R. et al., Chromatographia, 1991, 31 (11/12),255-256: Human bile was added at a concentration of 0.3% (v/v). Freshlystreaked cultures were examined for growth after 24 and 48 h.

Strain 35624 was capable of growth in the presence of physiologicallyrelevant human bile (0.3% (v/v)).

Growth of the strains was examined in the presence of individualconjugated and deconjugated bile acids. Under physiological conditionsbile acids are often found as sodium salts. The strains were screenedfor growth on TPY agar containing the conjugated and deconjugated sodiumsalts of each of the following bile acids.

(a) conjugated form: glycocholic acid (GCA); glycodeoxycholic acid(GDCA); and glycochenodeoxycholic acid (GCDCA);

(b) deconjugated form: lithocholic acid (LCA); chenodeoxycholic acid(CDCA); deoxycholic acid (DCA) and cholic acid (CA). For each bile acidconcentrations of 1, 3 and 4 mM were used. Growth was recorded after 24and 48 h anaerobic incubation.

The five strains studied grew on agar medium supplemented with 5 mM GCAand GCDCA and on agar medium supplemented with 1 mM GDCA as shown inTable 4. Strain 35624 was resistant to concentrations of 5 mM LCA (datanot shown) and strains 35612 and 35624 were capable of growth atconcentrations of 5 mM CA as shown in Table 5. No growth was observed inthe presence of 1 mM CDCA (data not shown). TABLE 4 Growth ofBifidobacterium sp. isolates in the presence of glycine-conjugated bileacids BILE ACIDS (mM) GCDCA GDCA GCA STRAIN 0 1 3 5 0 1 3 5 0 1 3 535612 + + + + + + + + + + + + 35624 + + + + + + + + + + + +35652 + + + + + + + + + + + + 35658 + + + + + + + + + + + +35687 + + + + + + + + + + + +−, no growth;+, confluent growthGCDCA, glycochenodeoxycholic acid,GDCA, glycodeoxycholic acid;CGA, glycocholic acid.

TABLE 5 Growth of Bifidobacterium sp. isolates in the presence ofunconjugated cholic acid (CA) CHOLIC ACID (mM) STRAIN 0 1 3 535612 + + + + 35624 + + + + 35652 + + − − 35658 + + − − 35687 + + − −−, no growth; +, confluent growth

EXAMPLE 4 Antimicrobial Activity

Bifidobacterium species exert inhibitory effects on other bacteria byexcluding long term colonisation by invasive pathogens. Theirantagonistic activity is due to the production of acetic and lactic acidthough fermentation (Scardovi, V. (1986) Bifidobacterium in Bergey'sManual of systemic bacteriology, Vol. 2. Eds. Sheath, P. H., Main, N.S., Sharpe, M. and Holdt, J. G., Williams and Wilkins Publishers,Baltimore Md., p 1418). Very few reports exist on the production ofantimicrobial compounds other than acids (Anand, S. K. et al. Cult.Dairy Prods. 1985;J. 2, 21-23). Bacteriocins and other compounds mayinfluence the survival of a bacterium in an ecological niche and allowthem to effectively dominate fermenting ecosystems. Such a feature is agood trait for a probiotic strain.

The inhibitory spectra of various bifidobacterial strains was determinedby the method of Tagg et al. (Tagg. J. R. et al. Bacteriol. Rev. 1976;40, 722-756). Cell free supernatant was assayed for inhibitory activityagainst a wide range of Gram positive and Gram negative micro-organisms.Overlays of each indicator were prepared on agar plates and allowed todry. Spots (5 ml) of cell free supernatant were placed on the seededplates, allowed to dry and the plates were incubated overnight.

It was observed that the strains were inhibitory to a wide range ofStaphylococcus, Pseudomonas, Coliform and Bacillus sp. when tested onTPY medium. Zones of inhibition of up to 4.4 mm were recorded againstPseudomonas and Staphylococcus and up to 7.0 mm surrounding Bacillus sp.as shown in Tables 6 and 7. However, when the deferred assays wereperformed on buffered TPY medium zones of inhibition were not observedagainst any indicator strain. Therefore, inhibition appeared to be,solely due to the presence of acid produced by the bifidobacteria. TABLE6 Inhibition of Staphylococcus strains by Bifidobacterium sp. onunbuffered medium* B. longum 1 B. longum 9 B. longum 10 63 35612 3562435652 35658 35675 35678 35687 S. aureus MHS 1.5 2 1.5 3.5 1.5 1 2 2 12.5 1.5 S. aureus HC 1.5 1.5 2 2.5 2 1.5 2.5 2 1.5 1.5 2 S. aureus 7711.5 3 1.5 3 2 2 2.5 2 3 2 3.5 S. aureus 949 2 3.5 2.5 2 3 3.5 3 2.5 3.53.5 2.5 S. aureus 1018 1 3.5 1.5 1.5 2 3.5 1 3 3.5 2.5 2 S. aureus 15021.5 3.5 1 2 2.5 2.5 1.5 3 4 2.5 1.5 S. aureus 1505 3 4 3 2.5 2.5 3 2.54.5 5.5 5 5.5 S. aureus 1511 1 3.5 2 1.5 2 2.5 3 3.5 4 2.5 3 S. aureus1522 1.5 3 2.5 1 2.5 1.5 2.5 2.5 3.5 3.5 3 S. aureus 1499 1.5 3.5 1.51.5 2 2 3 2 3.5 3.5 1.5 S. aureus 1963 2 3 2 2.5 3.5 3.5 3.5 3.5 2.5 32.5 S. aureus PRMM 1 3.5 1 1.5 1 3.5 2 2 3 2 2.5 S. albus 1 2 1.5 1 22.5 2 1.5 2 1.5 1 S. camosus 1 1.5 2 2.5 2.5 2.5 2 2.5 2 1.5 1*values given are radii of inhibition zones in mm (distance from edge ofproducer colony to the edge of zone of inhibition)

TABLE 7 Inhibition of Pseudomonas and Bacillus strains byBifidobacterium sp. on unbuffered medium* B. longum 1 B. longum 9 B.longum 10 63 35612 35624 35652 35658 35675 35678 35687 P. fluorescens HC1 2.5 1.5 1 1.5 2 3 2 1.5 2 2.5 P. fluorescens MHP 1.5 4.5 3.5 2 2.5 3.52.5 2.5 3.5 2 4 P. fluorescens DW 1.5 4 4 3.5 2.5 3.5 2.5 4.5 5.5 3.5 5B. cereus 3 3 5 3 4 4 3.5 5 6 4.5 5.5 B. subtilis 2 2.5 5 2 3 6 3 6 7 36 B. circulans 1 2 4 1.5 2.5 1.5 5 3.5 4.5 2 4.5 B. thuringensis 2.5 3.55 3 3.5 4.5 4 5.5 6.5 4.5 5.5*values given are radii of inhibition zones in mm (distance from edge ofproducer colony to the edge of zone of inhibition)

EXAMPLE 5 Murine Feeding Trial to Investigate the Ability ofLactobacillus salivarius subsp. Salivarius UCC 118 and Bifidobacteriumlongum infantis 35624 to Alleviate the Symptoms of Inflammatory BowelDisease (IBD)

Background

A number of mouse models have recently been generated by either geneticor immunological means to study the mechanisms of IBD. One of thesemodels involves the transfer of spleen or lymph node-derived CD4⁺Tlymphocytes from normal mice into severe combined immunodeficientrecipient mice (SCID). It has been demonstrated that mice who receivepurified CD4⁺, CD45RB^(high) T cells develop a wasting diseasecharacterised by chronic intestinal inflammation which is more severe inthe colon. In this study a control group of SCID mice was injected withCD4⁺CD45RB^(high) and the mice developed a progressive wasting diseaseincluding hunched over appearance, piloerection of the coat, diarrhoea,weight loss and macro and microscopic colon damage. A feeding trail wasset up administering UCC 118 and strain 35624 (also referred to hereinas UCC 35624) to determine if the symptoms of IBD could be modified inthis model.

Bacterial Strains

Lactobacillus salivarius subsp. Salivarius UCC 118 and Bifidobacteriumlongum infantis UCC 35624 were isolated from the ileal-caecal region ofan adult human as described in Example 1. In this example, spontaneousrifampicin and streptomycin resistant derivatives of the strains weregenerated by plating cells, previously grown overnight and subsequentlywashed in quarter strength Ringer's solution on MRS and TPY agarcontaining 50 μg/ml rifampicin (Sigma) respectively and MRS containing400 μg/ml streptomycin (Sigma). Plates were incubated for 2 days at 37°C. anaerobically. The resulting antibiotic resistant derivatives weredetermined to be otherwise phenotypically similar to the parent strain.This selectable trait enabled the strains to be readily enumeratedfollowing gut transit.

Animals and Maintenance

Donor mice (C57BL/6×BALB/c) F1 were purchased from Simosen Laboratories(Gilroy, Calif.) and maintained at the University of California—LosAngeles vivarium in ventilated cage racks (Thoren caging systems,Hazelton, Pa.) under specific pathogen free (SPF) conditions. CB-17 SCIDmice were bred in ventilated cage racks originally obtained from theUniversity of California—Los Angeles SCID core facility. The mice werereduced flora (RF) mice rather than germ free and acting as therecipient mice (Aranda R. et al. J. of Immunol. 1997; 158(7),3464-3473).

Eight week old, female CB17 (SCID) mice were housed in pairs in filtertop cages in ventilated racks. The mice were divided into four groupsGroup A: consumed 10% skim milk, control; Group B: consumedLactobacillus salivarius UCC 118, Group C: consumed Lactobacillussalivarius UCC 118 and Bifidobacterium longum UCC 35624 9 (1:1 ratio);Group D: consumed Bifidobacterium longum UCC 35624. UCC 118 and UCC35624 which were grown overnight in MRS broth and MRS broth supplementedwith 0.05% cysteine (Sigma) respectively, were washed in PBS,resuspended in skim milk (10% (v/v)) and administered in the otherwisesterile drinking water (PBS). The mice in each respective group received2.55×10⁸ cfu/ml of UCC 118 and 2.35×10⁸ cfu/ml of UCC 35624 daily forthe duration of the feeding period. Control mice received sterile milkdiluted in sterile phosphate buffered saline (PBS) and were maintainedunder identical conditions as the test group.

Experimental Design

All CB 17 mice were administered their respective feed according totheir grouping for 2 days prior to injection with the CD4⁺CD45RB^(high)cells. The sorted donor lymphocytes (3-4×10⁵) were represented in 200 μlof sterile PBS and injected i.p. into the recipient CB-17 SCID mice. Allmice were weighed initially, then twice weekly thereafter. They wereobserved for clinical signs of illness: hunched over appearance,piloerection of the coat and diarrhoea.

Evaluation of the Effects of the Administered Probiotics on the Numbersof Indigenous Bacteria Culturable from Mouse Faeces.

The influence exerted by the administered UCC 118 and UCC 35624 wheneither administered alone or in combination with each other, on themicroflora of the CB-17 SCID murine gut was investigated. Faecal sampleswere collected from each mouse weekly, weighed and resuspended in 10 mlPBS. The samples were then serially diluted in PBS and either pourplated or spread plated in appropriate dilutions on appropriate media induplicate. The following bacterial groups were enumerated: lactobacilli;bifidobacteria; enterococci; bacteroides and coliforms. The selectivemedia used were; de Mann Rogosa & Sharpe (MRS) agar, MRS agarsupplemented with 0.2% lithium chloride (BDH), 0.3% sodium propionate(Fluke chemie), 0.5% cysteine hydrochloride (Sigma), and 5% sheep'sblood; Slanetz and Bartley agar; Wilkins and Chalgren agar supplementedwith anaerobic supplement SR 108 and 5% horse blood; and Violet Red BileAgar. (All Oxoid unless otherwise stated). VRBA and Slanetz and Bartleyplates were incubated aerobically for 24 and 45 h respectively. Allother plates were incubated anaerobically for 48 h at 37° C.

Enumeration of Culturable Indigenous Flora from Specific Segments of theCB.17 SCID Murine G.I.T.

After the feeding period all mice were sacrificed and dissected.Segments of the ileal-caecal region, small intestine, and the largeintestine were removed. A peripheral lymph node (PLN), mesenteric lymphnode (MLN) and a piece of the spleen were also taken. All tissues wereweighed before being resuspended in 10 ml of PBS. Samples were thenhomogenised and serially diluted in PBS and either spread plated or pourplated in appropriate dilutions on appropriate media in duplicate. Thebacterial groups were enumerated the same as those enumerated in thefaecal analysis and samples were incubated as described previously.

Preparation of Intraepithelial and Lamniinapropria Lymphocytes

The isolation of the mucosal lymphocytes was carried out according tothe method of Aranda, R. et al ((1997) supra).

Flow Cytometic Analysis of Lymphocyte Populations.

The analysis was conducted as described by Aranda, R. et al. ((1997)supra)

Preparation of Tissue for Histopathological Analysis

Tissue samples were taken from the small intestine, large intestine, andileal caecal region and fixed in 10% formalin. The procedure was asdescribed in Aranda, R. et al. ((1997) supra).

It was observed from the experiment carried out that, consistent withprevious results, the SCID mice reconstituted with CD4⁺CD45RB^(high) Tlymphocytes and consuming skim milk alone (control) developed aprogressive wasting disease, identified by their significant weightloss. Disease became apparent at about 2 and a half to three weeks andthe sick mice characteristically manifested a hunched over appearance,piloerection of their coat, and loose stool. One of the mice in thecontrol group (mouse 4) died after 25 days and mice 1, 2, 3 and 5 showeda −20%, 25%, 21% and −35% percentage weight change respectively asdepicted in FIGS. 3 and 4.

CB-17 SCID mice consuming UCC 118 alone gave a similar result as thecontrols with the characteristic weight loss. Mouse 3 died after 14days, and mice 4, 5 and 6 showed a −15%, −25% and −28% percentage weightchange respectively (data not shown). The mice consuming a combinationof UCC 118 and UCC 35624 were found to have a marked improvement on thecontrol mice. These mice did not lose as much weight as the control miceover the feeding period. Even after 35 days three of the mice in thisgroup showed little percentage weight change. (FIGS. 5 and 6). Two ofthe mice in this group showed a weight loss only after about 30 dayswhereas control mice showed weight loss at 14 days (FIGS. 3 and 4).

Mice consuming UCC 35624 alone appeared in good health and again weightloss when compared to the controls was considerably less (FIGS. 7 and8). It can be concluded therefore that consumption of UCC 35624 eitheralone or in combination with UCC 118 alleviates the symptoms ofinflammatory bowel disease.

Table 8 is a summary of experimental data for the study on the treatmentof CD45RB colitis induced CB17 and SCID mice with a cocktail of UCC 118and UCC 35624.

It was found in the studies that the mice were successfullyreconstituted with lymphocytes and lymphocytes having been derived fromthe donor model (data not shown). TABLE 8 Treatment of CD45RB colitisinduced CB 17 SCID mice with a cocktail of Lactobacillus salivarius UCC118 and Bifidobacteria. Mouse 1 Untreated Mouse 2 Untreated Mouse 3Mouse 4 Mouse 5 Mouse 6 (RB hi cells + skimmed (RB hi cells + skimmedCocktail Cocktail Cocktail Cocktail Organ milk) milk) Treated TreatedTreated Treated % weight loss 31.25 27.74 14.50 14.05 21.88 11.18 Finallooks ill very ill very healthy slightly ill healthy healthy AppearanceStool very mushy very mushy mushy solid semi solid semi solid AppearanceColon thickened very slightly slightproximal slightly slightproximalAppearance thickened thickened thickening thickened thickening No. SIEL100,000 200,000 0 0 512,000 28,000 No. LIEL 25,000 72,000 100,000 50,000384,000 96,000 No. SLPL 200,000 100,000 264,000 200,000 640,000 104,000No. LLPL 96,000 256,000 160,000 160,000 256,000 160,000 No. MLN 0 N/A81,900 N/A 28,800 N/A No. PLN 0 192,000 0 120,000 64,000 0 Spleen #960,000 512,000 640,000 640,000 512,000 6,400,000 Lymphos. CD3+/H-2 Kb+Flow correction % No. SIEL 62,000 114,000 0 0 450,560 17,920 No. LIEL21,250 48,960 74,800 38,000 345,600 65,280 No. SLPL 74,000 42,000158,400 136,000 384,000 66,460 No. LLPL 67,200 161,280 115,200 108,000184,320 108,800 No. MLN 0 N/A 130,00 N/A 64,000 N/A No. PLN 0 126,720 087,600 54,400 0 Spleen 518,400 102,400 211,200 307,200 230,400 4,480,000UCC 118 bacterial counts (per biopsy) post mortem SI 0 0 1,200 0 0 0 LI0 0 >30,000 >30,000 100 11,600 Caecum 00 >30,000 >30,000 >30,000 >30,000 Spleen 0 0 0 1,350 0 0 ColonPathological Scoring A (0-3) — 1.0 1.0 2.0 — — B (0-2) — 1.5 1.0 1.0 — —C (0-3) — 2.5 1.0 2.0 — — D (0-3) — 2.0 3.0 3.0 — — E (1-3) — 1.0 1.02.0 — Remarks Total Score — 8.0 7.0 10.0 — —A: Degree of inflammatory infiltrate;B: Mucin depletion;C: Epithelia hyperplasia;D: No. of TEL in the crypts;E: No. of inflammatory foci per high power fields

EXAMPLE 7 In Vitro Studies to Examine the Immune Perception ofBifidobacterium longum infantis

Overnight washed cultures of Bifidobacteria were incubated with humanperipheral blood mononuclear cells (PBMCs) from both healthy volunteers(n=9) and patients suffering from inflammatory bowel disease (n=5).Production of the proinflammatory cytokine tumour necrosis factor α(TNFα) was measured by ELISA in seventy two hour culture supernatants.Co-incubation of Bifidobacterium longum infantis with human PBMCs didnot result in the stimulation of TNFα production (FIG. 9). Thus,exposure of the systemic immune system to this bacterium does induce aninflammatory response.

In order to assess the immune perception of Bifidobacterium longuminfantis at mucosal surfaces, co-culturing of epithelial cells and PBMCswas performed in transwell chambers. Briefly, an epithelial cellmonolayer air was grown in the upper chamber and PBMCs were incubated inthe lower compartment. These were separated from each other by a porousmembrane which allowed the passage of soluble mediators between the twocompartments but did not allow cell-cell contact. Using this model, theproduction of TNFα and interleukin-8 (IL-8) was measured in the presenceand absence of Bifidobacterium longum infantis in the PBMC compartment.Co-culture of epithelial cells, PBMCs and Bifidobacterium longuminfantis resulted in significant suppression of TNFα and IL-8 production(FIG. 10). Thus, a tri-cellular network involving epithelial cells,PBMCs and Bifidobacterium longum infantis results in suppression ofproinflammatory cytokine production.

EXAMPLE 8 In Vivo Anti-Inflammatory Activity of Bifidobacterium longuminfantis

Bifidobacterium longum infantis (1×10⁹ cells per day) was consumed by 18healthy humans in a fermented milk (yoghurt) product for three weeks.Serum was collected for cytokine analysis pre and post consumption ofthis probiotic strain. Faecal samples were obtained for microbiologicalanalysis.

Considerable modification of peripheral blood cytokine levels wereobserved in this feeding study. Serum soluble Interleukin-6 receptor(sIL-6R, p=0.007), Interferon-γ (IFNγ, p=0.041) and IL-8 (p=0.004)levels were significantly reduced following consumption of thisprobiotic strain (FIG. 11). No alteration in serum TNFα andInterleukin-1 receptor antagonist (IL-1RA) levels were observed (FIG.12). Bifidobacterium longum infantis was detected at approximately 1×10⁵colony forming units per gram of faecal matter over the course of thisfeeding study.

Targeted in vitro selection criteria reflecting the complex interactionsof the GI environment allow for the identification of probiotic strainscapable of functioning effectively when reintroduced into thatenvironment. Using the selection criteria outlined above, the probioticbacteria Bifidobacterium longum infantis has demonstrableimmunomodulating properties in vitro. Following consumption by SCID miceand human volunteers, significant modification of systemic immuneparameters was noted. Thus, the use of Bifidobacterium longum infantisas a biotherapeutic agent in the treatment of immune mediated diseasesis warranted.

EXAMPLE 9 Measurement of TNFα in Bifidobacterium longum infantis UCC35624 Cell Free Supernatant

Overnight cultures of Bifidobacterium longum infantis were centrifugedand the cell-free culture supernatant was examined for the presence ofcytokine inhibitors. Cell free supernatants were incubated with humanTNFα for 20 minutes at 37° C. TNFα levels were quantified thereafter byELISA. Following exposure to the Bifidobacteria supernatant, TNFα levelswere significantly reduced (FIG. 13). Thus, Bifidobacterium longuminfantis UCC35624 secretes a factor that antagonises TNFα activity.Production of this factor by Bifidobacterium longum infantis at thesurface of the gastrointestinal tract, in vivo, would significantlyrestrict the host inflammatory response.

This indicates that the antagonism of TNFα also occurs at a molecularlevel due to a soluble factor released by UCC 35624

Inflammation

Inflammation is the term used to describe the local accumulation offluid, plasma proteins and white blood cells at a site that hassustained physical damage, infection or where there is an ongoing immuneresponse. Control of the inflammatory response is exerted on a number oflevels (for review see Henderson B., and Wilson M. 1998. In“Bacteria-Cytokine interactions in health and disease. Portland Press,79-130). The controlling factors include cytokines, hormones (e.g.hydrocortisone), prostaglandins, reactive intermediates andleukotrienes. Cytokines are low molecular weight biologically activeproteins that are involved in the generation and control ofimmunological and inflammatory responses, while also regulatingdevelopment, tissue repair and haematopoiesis. They provide a means ofcommunication between leukocytes themselves and also with other celltypes. Most cytokines are pleiotrophic and express multiple biologicallyoverlapping activities. Cytokine cascades and networks control theinflammatory response rather than the action of a particular cytokine ona particular cell type (Arai K I, et al., Annu Rev Biochem1990;59:783-836). Waning of the inflammatory response results in lowerconcentrations of the appropriate activating signals and otherinflammatory mediators leading to the cessation of the inflammatoryresponse. TNFα is a pivotal proinflammatory cytokine as it initiates acascade of cytokines and biological effects resulting in theinflammatory state. Therefore, agents which inhibit TNFα are currentlybeing used for the treatment of inflammatory diseases, e.g. infliximab.

Pro-inflammatory cytokines are thought to play a major role in thepathogenesis of many inflammatory diseases, including inflammatory boweldisease (IBD). Current therapies for treating IBD are aimed at reducingthe levels of these pro-inflammatory cytokines, including IL-8 and TNFα.It has been suggested that such therapies may also play a significantrole in the treatment of systemic inflammatory diseases such asrheumatoid arthritis. Humans fed with yoghurt containing Bifidobacteriumlongum infantis UCC35624 have shown marked decreases in their systemiclevels of IL-8. This strain may therefore have potential application inthe treatment of a range of inflammatory diseases, particularly if usedin combination with current anti-inflammatory therapies, such asnon-steroid anti-inflammatory drugs (NSAIDs) or Infliximab.

Diarrhoeal Disease.

The barrier function of the intestinal epithelium can be diminishedduring nervous (acetylcholine) and immune (histamine) mediatedsecretion. Certain bacterial toxins may also induce Ca2⁺ and PKCdependent secretion and thereby can disturb the epithelial barrier(Ganguly N K and Kaur T. Indian J Med Res 1996;104:2837, Groot J A. VetQ 1998;20(S3):45-9). Several studies have examined the prevention andtreatment of diarrhoea using probiotic bacteria. Prospective studieshave demonstrated the efficacy of lactic acid bacteria administrationfor both prophylactic and therapeutic use against diarrhoea inpre-mature infants, new borns, children (Isolauri E, et al., Dig Dis SciDecember 1994;39(12):2595-600) and in the treatment of antibioticrelated diarrhoea (Siitonen S, et al., Ann Med February 1990;22(1):57-9)and traveller's diarrhoea (Oksanen P J, et al., Ann Med February1990;22(1):53-6).

We have examined consumption of Bifidobacterium longum infantis UCC35624 by SCID mice. It was found that inflammatory activity wassignificantly attenuated and mice consuming Bifidobacterium longuminfantis UCC 35624 retained solid stools while control mice sufferedfrom diarrhoea. FIGS. 14 and 15 illustrate the lower intestine oftreated and untreated SCID mice. The lower intestine shown includes thecaecum 2, intestine 3 and anus 5. In FIG. 14 the mice were treated withBifidobacterium longum infantis UCC 35624 and it is apparent that solidstools 4 have been retained in the intestine. In comparison FIG. 15shows the untreated mouse intestine 3, characteristically inflamed. Nowater absorption has occurred so that no solid stools are retainedresulting in diarrhoea.

The anti-diarrhoeal effect observed may be related to theanti-inflammatory activity, possibly mediated via cAMP modulation.Cyclic AMP-dependent C1-secretion is the major secretory pathway in thehuman intestine (Brzuszczak I M, et al., J. Gastroenterol. Hepatol.1996;11(9):804-10). It can be inferred that the anti-diarrhoeal effectof Bifidobacterium longum infantis UCC 35624 is not restricted just todiarrhoea resulting from gastrointestinal inflammation, but can beapplied to the general treatment of diarrhoeal disease.

Autoimmune Disease

The immune system has a large repertoire of specificities expressed by Band T cells. Some of these specificities will be directed toself-components. Self-recognition is normally controlled by clonaldeletion and inactivation of self-reactive lymphocytes. However, thereis a constant background of autoimmunity with antibodies to manyproteins being found in serum. A breakdown in the self-nonselfrecognition system results in autoimmunity. When autoimmune disease doesoccur, the resulting immune response damages the tissue bearing theoffending antigen. Immune complex deposition, type II hypersensitivityand cell-mediated reactions are the most important mechanisms by whichimmunopathological damage occurs. Examples of autoimmune diseasesinclude, but are not limited to, systemic lupus erythematosus,rheumatoid arthritis, insulin dependent diabetes mellitus, myastheniagravis and pernicious anaemia. Bifidobacterium longum infantis andLactobacillus salivarius subsp. salivarius are immunomodulatorybacteria. Thus, consumption either as single components or incombination of these bacteria by patients suffering from autoimmunedisease may restrict organ damage and help restore normal bodyhomeostasis.

Inflammation and Cancer

The production of multifunctional cytokines across a wide spectrum oftumour types suggests that significant inflammatory responses areongoing in patients with cancer. It is currently unclear what protectiveeffect this response has against the growth and development of tumourcells in vivo. However, these inflammatory responses could adverselyaffect the tumour bearing host. Complex cytokine interactions areinvolved in the regulation of cytokine production and cell proliferationwithin tumour and normal tissues (McGee, D W, et al., ImmunologySeptember 1995;86(1):6-11, Wu S, et al., Gynecol Oncol April1994;53(1):59-63). It has long been recognised that weight loss(cachexia) is the single most common cause of death in patients withcancer (Inagaki J, et al., Cancer February 1974;33(2):568-73) andinitial malnutrition indicates a poor prognosis (Van Eys J. Nutr RevDecember 1982;40(12):353-9). For a tumour to grow and spread it mustinduce the formation of new blood vessels and degrade the extracellularmatrix. The inflammatory response may have significant roles to play inthe above mechanisms, thus contributing to the decline of the host andprogression of the tumour. Due to the anti-inflammatory nature of thesebacterial strains they may reduce the rate of malignant celltransformation. Furthermore, intestinal bacteria can produce, fromdietary compounds, substances with genotoxic, carcinogenic andtumour-promoting activity and gut bacteria can activate pro-carcinogensto DNA reactive agents (Rowland I. R. (1995). Toxicology of the colon:role of the intestinal microflora. In: Gibson G. R. (ed). Human colonicbacteria: role in nutrition, physiology and pathology, pp 155-174. BocaRaton CRC Press). In general, species of Bifidobacteria andLactobacillus have low activities of xenobiotic metabolising enzymescompared to other populations within the gut such as bacteroides,eubacteria and clostridia (Saito Y., et al., Microb. Ecol. Health Dis.,1992;5, 105-110). Therefore, increasing the number of lactic acidbacteria in the gut could beneficially modify the levels of theseenzymes.

Prebiotics

The introduction of probiotic organisms is accomplished by the ingestionof the microorganism in a suitable carrier. It would be advantageous toprovide a medium that would promote the growth of these probioticstrains in the large bowel. The addition of one or moreoligosaccharides, polysaccharides, or other prebiotics enhances thegrowth of lactic acid bacteria in the gastrointestinal tract (Gibson, GR. Br. J. Nutr. 1998;80 (4):S209-12). Prebiotics refers to anynon-viable food component that is specifically fermented in the colon byindigenous bacteria thought to be of positive value, e.g.bifidobacteria, lactobacilli. Types of prebiotics may include thosewhich contain fructose, xylose, soya, galactose, glucose and mannose.The combined administration of a probiotic strain with one or moreprebiotic compounds may enhance the growth of the administered probioticin vivo resulting in a more pronounced health benefit, and is termedsynbiotic.

Other Active Ingredients

It will be appreciated that the Bifidobacterium may be administeredprophylactically or as a method of treatment either on its own or withother probiotic and/or prebiotic materials as described above. Inaddition, the bacteria may be used as part of a prophylactic ortreatment regime using other active materials such as those used fortreating inflammation or other disorders, especially those of thegastrointestinal tract. Such combinations may be administered in asingle formulation or as separate formulations administered at the sameor different times and using the same or different routes ofadministration.

The invention is not limited to the embodiments hereinbefore describedwhich may be varied in detail.

55-129. (canceled)
 130. A biologically pure culture of a strain ofBifidobacterium isolated from a resected and washed humangastrointestinal tract, wherein the strain of Bifidobacterium secretes afactor that antagonizes the activity of at least one pro-inflammatorycytokine.
 131. The biologically pure culture of a strain ofBifidobacterium according to claim 130, herein the pro-inflammatorycytokine is TNF-α.
 132. The biologically pure culture of a strain ofBifidobacterium according to claim 130, wherein the pro-inflammatorycytokine is IL-8.
 133. The biologically pure culture of a strain ofBifidobacterium according to claim 130, wherein the strain ofBifidobacterium is bile resistant and grows in the presence ofphysiologically relevant human bile.
 134. The biologically pure cultureof a strain of Bifidobacterium according to claim 130, wherein thestrain of Bifidobacterium is significantly immunomodulatory followingoral consumption in humans, as evidenced by a decrease in systemiclevels of at least one pro-inflammatory cytokine in humans.
 135. Thebiologically pure culture of a strain of Bifidobacterium according toclaim 134, wherein the pro-inflammatory cytokine is TNF-α.
 136. Thebiologically pure culture of a strain of Bifidobacterium according toclaim 134, wherein the pro-inflammatory cytokine is IL-8.
 137. Thebiologically pure culture of a strain of Bifidobacterium according toclaim 134, wherein the pro-inflammatory cytokine is sIL-6R.
 138. Thebiologically pure culture of a strain of Bifidobacterium according toclaim 134, wherein the pro-inflammatory cytokine is IFNγ.
 139. Thebiologically pure culture of a strain of Bifidobacterium according toclaim 134, wherein the immunomodulatory effect is an increase in ananti-inflammatory cytokine; a decrease in a pro-inflammatory cytokine;an increase in an anti-inflammatory cytokine and a decrease in apro-inflammatory cytokine; or production of a high anti-inflammatorycytokine to pro-inflammatory cytokine ratio in the human following oralconsumption.
 140. The biologically pure culture of a strain ofBifidobacterium according to claim 134, wherein the strain inhibits thegrowth of Gram positive bacteria, Gram negative bacteria, or both. 141.The biologically pure culture of a strain of Bifidobacterium accordingto claim 134, wherein the strain inhibits the growth of Staphylcoccusspp., Pseudomonas spp., Coliform spp., Bacillus spp., or a combinationthereof.
 142. The biologically pure culture of a strain according toclaim 134, wherein the strain of Bifidobacterium comprisesBifidobacterium longum infantis.
 143. The biologically pure culture of astrain of Bifidobacterium according to claim 134, wherein the strain isBifidobacterium longum infantis UCC35624 or a mutant or a variantthereof.
 144. The biologically pure culture of a strain according toclaim 143, wherein the strain of Bifidobacterium comprisesBifidobacterium longum infantis UCC35624.
 145. The biologically pureculture of a strain of Bifidobacterium according to claim 143, whereinthe mutant is a genetically modified mutant.
 146. The biologically pureculture of a strain of Bifidobacterium according to claim 143, whereinthe variant is a naturally occurring variant.
 147. The biologically pureculture of a strain of Bifidobacterium according to claim 134, whereinthe strain is in the form of viable cells.
 148. The biologically pureculture of a strain of Bifidobacterium according to claim 134, whereinthe strain is in the form of non-viable cells.
 149. The biologicallypure culture of a strain of Bifidobacterium according to claim 134,wherein the strain of Bifidobacterium is immunomodulatory tosubstantially the same extent as Bifidobacterium longum infantisUCC35624.
 150. A formulation comprising the biologically pure culture ofa strain of Bifidobacterium isolated from a resected and washed humangastrointestinal tract, wherein the strain of Bifidobacterium secretes afactor that antagonizes the activity of at least one pro-inflammatorycytokine.
 151. The formulation of claim 150 comprising two or morestrains of Bifidobacterium.
 152. The formulation of claim 150, whereinthe strain of Bifidobacterium is significantly immunomodulatoryfollowing oral consumption in humans, as evidenced by a decrease insystemic levels of at least one pro-inflammatory cytokine in humans.153. The formulation of claim 150, further comprising an additionalprobiotic material or a prebiotic material.
 154. The formulation ofclaim 150, further comprising a strain of Lactobacillus salivarius. 155.The formulation of claim 154, wherein the strain of Lactobacillussalivarius is in the form of viable cells.
 156. The formulation of claim154, wherein the strain of Lactobacillus salivarius is in the form ofnon-viable cells.
 157. The formulation of claim 154, wherein the strainof Lactobacillus salivarius is isolated from a resected and washed humangastrointestinal tract and is significantly immunomodulatory followingoral consumption in humans.
 158. The formulation of claim 154, whereinthe strain of Lactobacillus salivarius inhibits the growth of Grampositive bacteria, Gram negative bacteria, or both.
 159. The formulationof claim 154, wherein the strain of Lactobacillus salivarius isLactobacillus salivarius strain UCC 118 or a mutant or a variantthereof.
 160. The formulation of claim 159, wherein the mutant is agenetically modified mutant.
 161. The formulation of claim 159, whereinthe variant is a naturally occurring variant.
 162. The formulation ofclaim 150, further comprising an ingestible carrier.
 163. Theformulation of claim 162, wherein the ingestible carrier is apharmaceutically acceptable carrier.
 164. The formulation of claim 163,wherein the pharmaceutically acceptable carrier is in the form of acapsule, a tablet, or a powder.
 165. The formulation of claim 162,wherein the ingestible carrier is a food product.
 166. The formulationof claim 165, wherein the food product is acidified milk, a yogurt, afrozen yogurt, a milk powder, a milk concentrate, a cheese spread, adressing, or a beverage.
 167. The formulation of claim 150, furthercomprising a protein, a peptide, a lipid, a carbohydrate, a vitamin, amineral, or a trace element.
 168. The formulation of claim 167, whereinthe protein or the peptide is rich in glutamine, glutamate, or both.169. The formulation of claim 150, wherein the Bifidobacterium ispresent at more than 10⁶ cfu per gram of the formulation.
 170. Theformulation of claim 150, further comprising an adjuvant.
 171. Theformulation of claim 150, further comprising a bacterial component. 172.The formulation of claim 150, further comprising a drug entity.
 173. Theformulation of claim 150, further comprising a biological compound. 174.The formulation of claim 150, wherein the formulation is suitable fororal administration to a subject.
 175. A foodstuff comprising the strainof Bifidobacterium of claim
 134. 176. A foodstuff comprising theformulation of claim
 150. 177. A pharmaceutical composition comprisingthe Bifidobacterium strain of claim 134 and a pharmaceuticallyacceptable carrier.
 178. A pharmaceutical composition comprising theformulation of claim 150 and a pharmaceutically acceptable carrier. 179.The biologically pure culture of a strain of Bifidobacterium accordingto claim 134, wherein the strain is capable of reducing or inhibitingthe effects of inflammatory bowel disease in the presence ofphysiological concentrations of human bile and human gastric juice. 180.The biologically pure culture of a strain of Bifidobacterium accordingto claim 179, wherein the strain is capable of reducing or inhibitingthe effects of inflammatory bowel disease in the presence ofphysiological concentrations of human bile and human gastric juice, asevidenced by measuring a reversal of wasting disease induced in severecombined immunodeficient recipient mice (SCID) which have beenadministered purified CD4⁺, CD45RB^(high) T cells.
 181. The formulationof claim 150, wherein the formulation is capable of reducing orinhibiting the effects of inflammatory bowel disease in the presence ofphysiological concentrations of human bile and human gastric juice. 182.The formulation of claim 181, wherein the formulation is capable ofreducing or inhibiting the effects of inflammatory bowel disease in thepresence of physiological concentrations of human bile and human gastricjuice, as evidenced by measuring a reversal of wasting disease inducedin severe combined immunodeficient recipient mice (SCID) which have beenadministered purified CD4⁺, CD45RB^(high) T cells.